Heterologous Expression and Characterization of a Novel Exo-Polygalacturonase from Aspergillus fumigatus Af293 and Its Application in Juice Extraction

Exo-polygalacturonase (exo-PG) hydrolyzes pectin acids and liberates mono-galacturonate, which plays an important role in juice extraction, and has rarely been reported. Exo-PG (AfumExoPG28A) from Aspergillus fumigatus belongs to the glycoside hydrolase 28 family. In this study, its gene was cloned and the protein was expressed and secreted in Pichia pastoris with a maximal activity of 4.44 U/ml. The optimal temperature and pH of AfumExoPG28A were 55°C and 4.0, respectively. The enzyme exhibited activity over almost the entire acidic pH range (>20.0% activity at pH 2.5-6.5) and remained stable at pH 2.5–10.0 for 24 h. The Km and Vmax values of AfumExoPG28A were calculated by the substrate of polygalacturonic acid as 25.4 mg/ml and 23.6 U/mg, respectively. Addition of AfumExoPG28A (0.8 U/mg) increased the light transmittance and juice yield of plantain pulp by 11.7% and 9%, respectively. Combining AfumExoPG28A (0.8 U/mg) with an endo-PG (0.8 U/mg) from our laboratory, the enzymes increased the light transmittance and juice yield of plantain pulp by 45.7% and 10%, respectively. Thus, the enzyme’s potential value in juice production was revealed by the remarkable acidic properties and catalytic activity in fruit pulp.


Transformation and Heterologous Expression
Recombinant plasmid pPIC9K-AfumExoPG28A was extracted using a plasmid extraction kit. Next, 1 μg of recombinant plasmid linearized by Sac I was transformed into P. pastoris GS115 through the LiCl method. After preliminary screening on MD plates, the positive transformants were selected by 2.5 mg/ml G418 on YPD plates and confirmed by PCR using the primers 5´AOX (5´-GACTGGTTCCAATTGACAAGC-3´) and 3´AOX (5´-GCAAATGGCATTCTGACATCC-3´) .
Next, in a 250-ml flask, one transformant was selected and inoculated into 50 ml BMGY liquid medium, cultured at 30°C and 200 rpm. When the OD 600 reached 3.0, the cells were centrifuged at 2,000 ×g for 10 min at room temperature, and the supernatant was discarded. The cells were transferred to 25 ml BMMY liquid medium in a 250-ml flask and cultured at 30°C and 200 rpm. To induce the expression of AfumExoPG28A, 0.5% methanol was added in daily and the culture solution was sampled for enzyme production every 24 h. After 120 h of methanol induction, the culture solution was centrifuged at 4°C and 12,000 ×g. The supernatants were used as crude enzyme.

Purification and Determination
The 100 ml culture solution of recombinant protein was concentrated with a molecular weight cut-off of 10 kDa using an ultrafiltration device (Ireland). Then, 20 mM Tris-HCl buffer (pH 7.0) was used to replace the culture solution, and the final solution was stored at 4°C until further analysis. The protein concentration was determined by the Bradford method [19]. The homogeneity of the protein was checked via SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using 5% stacking gel and 10% separating gel) [20]. The purified recombinant protein was deglycosylated by Endo H (20 μl reaction system: 20 μg protein, 3 μl enzyme dosage, and 2 μl 10 × GlycoBuffer 3 in ddH 2 O 37°C for 3 h) and then analyzed by SDS-PAGE.

Enzyme Activity Assay
The activity of AfumExoPG28A was measured by the 3,5-dinitrosalicylic acid (DNS) method [21]. One unit of polygalacturonase activity is defined as the amount of enzyme required to release 1 mol of D-galacturonic acid per minute [22].

Determination of the Optimum Temperature and pH for AfumExoPG28A Activity and Stability
The optimal pH for AfumExoPG28A was determined at 50°C in the range of pH 2.5 to 7.0. The maximum enzyme activity was set as 100%, and enzyme activities at other pH values were normalized to this maximum value. The stability of AfumExoPG28A at different pH values was analyzed in different buffers (0.1 M citric acid-Na 2 HPO 4 for pH 3.0-7.0, 0.1 M Tris-HCl for pH 7.0-9.0, and 0.1 M Gly-NaOH for pH 9.0-10.0) at 4°C for 24 h. The residual activities were determined after 15 min treatment at 50°C and at optimal pH.
The optimum temperature of AfumExoPG28A was analyzed at 25-80°C under optimal pH conditions. The maximum enzyme activity was defined as 100%, and other enzyme activities were calculated as relative values. To explore its thermostability, the enzyme solution was incubated at 45, 50, and 55°C for 1 h, and the residual activity was measured every 15 min.

Determination of Kinetic Parameters and Substrate Specificity
To determine the kinetic parameters, different concentrations (0.20-1.0%) of PGA were used to measure the enzyme activity under optimal conditions (pH 4.0 and 55°C). The K m and V max values were calculated by the Lineweaver-Burk method. For the substrate specificity, 0.5% (w/v) PGA, 0.5% (w/v) citrus pectin, 0.5% (w/v) apple pectin, 0.5% (w/v) carboxymethyl cellulose, and 0.5% (w/v) xylan were used under optimal conditions. The enzyme activity of AfumExoPG28A with 0.5% (w/v) PGA was defined as 100%, and activities in other reactions were normalized to this value.

Effects of Metal Ions on AfumExoPG28A Activity
The presence of metal ions may affect enzyme activity in many ways. To determine the effects of metal ions on enzyme activity, the effects of 1 or 2 mM of Zn 2+ , K + , Ca 2+ , Mg 2+ , Ba 2+ , Na + , Mn 2+ , Cu 2+ , Co 2+ , and Fe 2+ on the catalytic activity of AfumExoPG28A were measured. The control value with no metal ions was defined as 100%, and activities in other reactions were normalized to this value.

TLC Analysis of Hydrolysis Products of PGA by AfumExoPG28A
The purified samples of AfumExoPG28A and 0.5% PGA with 0.1 M citric acid-Na 2 HPO 4 (pH 4.0) were incubated at 55°C for 0.25, 0.5, 1.0, 2.0, 8.0, and 12 h. The control sample did not contain AfumExoPG28A. The main hydrolysis products were analyzed by TLC to demonstrate the action mode of AfumExoPG28A using a previously reported method [23].

Use of AfumExoPG28A to Extract Fresh Plantain Juice
Fresh and disease-free fruits (plantain) were purchased at a local market. Next, 500 g fruit and 500 ml doubledistilled water were mixed and pulped. AfumExoPG28A (0.8 U/mg) was added to fresh pulp (25 g), and samples were incubated for 2 h at 55°C. Then, an endo-PG (0.8 U/mg) from our laboratory and AfumExoPG28A (0.8 U/ mg) were mixed together in fruit pulp under the same conditions. The same amount of pulp was also incubated without enzyme as a control. Then, fruit pulp in the control and experimental groups was centrifuged at 3,214 ×g for 10 min at 25°C. The supernatants were measured to determine the pH, juice extraction rate (1), and light transmittance (A 660 ) using previously reported methods [21].

Gene Cloning, Expression, and Purification of Recombinant Exo-PG
The 1,323-bp coding region of a putative exo-PG gene in the genome of A. fumigatus Af293 was obtained from the GenBank database. A putative, 21-residue signal peptide (Met1-Gly21) was predicted by SignalP4.1. In comparison with a functionally characterized protein (PgaII) [24], Asp 211 , Asp 231 , Asp 232 , and His 255 of AfumExoPG28A were expected to be the catalytic residues, and Arg 290 and Lys 292 of AfumExoPG28A were expected to be involved in substrate binding (Fig. 1). However, AfumExoPG28A shared the highest homology of only 27% with an experimentally verified exo-PG (TePG28a) from Talaromyces leycettanus [25] (GenBank Accession No. KY474617). Thus, AfumExoPG28A was a novel exo-PG, which is worthy of further study.
The recombinant vector pPIC9K-AfumExoPG28A was constructed without a putative signal peptide region and then successfully transformed to P. pastoris GS115. After 5 days of cultivation under the induction of methanol, a transformant whose PG activity reached 4.44 U/ml was obtained (Fig. 2). Heterologous expression is an efficient method to obtain novel enzymes. Many PG genes have been cloned and heterologously expressed in eukaryotic and prokaryotic cells with effective expression systems, such as PgaB from Aspergillus luchuensis [26,27], AnEPG from Aspergillus nidulans [28], and endoPG from Aspergillus aculeatus [29], etc., but only two exo-PG genes (exo-TePG28a from T. leycettanus [25] and RmGH28 from Rhodothermus marinus [30]) were expressed in P. pastoris and E. coli, respectively.
After a simple one-step procedure of ultrafiltration, the recombinant protein AfumExoPG28A with PG activity was purified to electrophoretic homogeneity, The purified AfumExoPG28A showed a molecular weight of approximately 62 kDa in SDS-PAGE (Fig. 3), which was higher than the calculated value of 46 kDa. After Endo H treatment, the molecular weight of de-glycosylated AfumExoPG28A matched the theoretical value. This confirmed the previously reported observation that heterologous proteins expressed in P. pastoris were generally excessively N-glycosylated [28,31].

Substrate Specificity and Determination of Kinetic Parameters of AfumExoPG28A
Purified AfumExoPG28A exhibited the highest specific activity towards polygalacturonic acid (PGA) (5.72 U/mg, 100%), followed by citrus pectin (1.44 U/mg, 25%) and apple pectin (0.21 U/mg, 4%), while showing almost no activity on CMC and xylan (Fig. 4). This result agreed with the fact that the methoxy groups can hinder the degradation of the pectin backbone by PGs [32]. Considering the above substrate specificity analysis results, AfumExoPG28A was characterized as a PG.

Effects of Temperature and pH on the Activity and Stability of AfumExoPG28A
The purified AfumExoPG28A showed maximal activity towards PGA at pH 4.0 (Fig. 6A), similar to those of most reported exo-PGs (pH 3.0-5.0) from filamentous fungi [35]. Interestingly, AfumExoPG28A exhibited PG   activity over almost the entire acidic pH range (>20.0% activity at pH 2.5-6.5) and remained stable at pH 2.5-10.0 for 24 h (Fig. 6B). PGs with high activity in acidic environments are widely used in fruit juice production and feed processing [2], but different fruit pulps have different pH values. In addition, feed can only be digested after staying in the acidic intestinal environment of animals for a long time [7]. Therefore, it is important to evaluate the pH range in which PGs are stable and active to determine their potential application value. Although most reported exo-PGs show catalytic activity at different pH values, a few remained active and stable under a wide pH range. For example, like AfumExoPG28A, the two exo-PGs from Penicillium sp. [36,37] were active in a wide pH  range, but they were only stable at pH 3.0-5.0 and pH 4.5-6.0, respectively. The optimal pH of exo-PG1 was 4.0, similar to that of AfumExoPG28A, but it remained stable only at pH 4.0-5.0 [38]. The optimal pH of exo-PG TePG28a from T. leycettanus was also similar to that of AfumExoPG28A, but it had little activity at pH 5.0 [25]. The two exo-PGs from Aspergillus sp. remained stable under acidic conditions, but they had almost no activity when the pH was lower than 4.0 [39,40]. Compared with these enzymes, AfumExoPG28A was both active and stable in a wide pH range, suggesting that it has great potential application value in agriculture and the food industry.
Temperature affects the spatial structure of the enzyme. High or low temperature reduce the catalytic efficiency of the enzyme, and too high temperatures will even cause irreversible protein denaturation [41]. The optimal temperature of AfumExoPG28A was 55°C, similar to most reported optimal temperatures for other PGs [36,37], and therefore it is a mesophilic pectinase (Fig. 6C). AfumExoPG28A was stable after incubation for 60 min at 45°C, and retained only 30% of its initial activity after incubation for 60 min at 50°C (Fig. 6D). The optimal temperatures of PGs are generally higher than their maximal temperatures of thermostability. The reason may be that higher temperatures facilitate the diffusion of substances, thus increasing the catalytic rate of the enzyme, while the combination of the enzyme and substrate can also improve the stability of the enzyme itself [42].

The Effects of Metal Ions on AfumExoPG28A Activity
Metal ions affect enzyme activity by binding to amino acid residues. Ca 2+ serves as an activator of many enzymes, but some studies showed that it could inhibit the activity of PGs [43]. The present study showed that AfumExoPG28A exhibited 73 ± 1.8% enzyme activity in the presence of 2 mM Ca 2+ (Table 1). Mn 2+ and Fe 2+ partially inhibited the activity of AfumExoPG28A, similar to Penicillium janthinellum VI2R3M [36]. Cu 2+ slightly increased the activity of AfumExoPG28A, similar to Aspergillus niger MTCC 478 [39]. The addition of other metal ions had little or no effect on enzyme activity, indicating that the activity of AfumExoPG28A might not depend on the presence of metal ions.

Analysis of Hydrolysis Products of PGA by AfumExoPG28A
The hydrolysis products of PGA by AfumExoPG28A were analyzed by thin-layer chromatography (TLC). As shown in Fig. 7, analysis of products at different time points (0-12 h) showed that no oligosaccharide was  produced, and the content of galacturonic acids increased with the extension of reaction time. This behavior was consistent with the previous finding that exo-PG can only hydrolyze the non-reducing terminal glucosyl residues of PGA and produce galacturonic acid monosaccharide products [2], indicating that AfumExoPG28A is an exo-PG.

Use of AfumExoPG28A for Fresh Plantain Juice Extraction
The cell walls of fruits and vegetables are rich in polysaccharide components such as pectin, cellulose, and hemicellulose, among which pectin can promote the cross-linking of polysaccharide components [2]. Therefore, the addition of pectinase to hydrolyze pectin in juice production can significantly improve the yield and clarity of juice [13]. Endo-PGs show high efficiency of pectin de-polymerization due to their ability to randomly degrade the PGA backbone in pectin molecules; hence, they have attracted wide attention in recent years [28]. While endo-PG is one of the most important pectinases, it had been found that the pectin de-polymerization efficiency could be significantly improved under the synergistic effect of exo-PG and endo-PG [25].
Based on the above results, we identified a novel exo-PG from A. fumigatus, indicating that the enzyme could have potential application value in fruit juice production. Next, plantain (Musa sp.) was selected as a research object because its pH (pH 4.5) is close to the optimal pH of AfumExoPG28A (pH 4.0). When the enzyme dosage was 0.8 U/mg, AfumExoPG28A increased the light transmittance and yield of plantain juice by 11.7% and 9.0%, respectively. Meanwhile, the endo-PG (0.8 U/mg) from our laboratory increased the light transmittance and yield of plantain juice by 31.1% and 9.0%, respectively. The combinations of AfumExoPG28A and endo-PG increased the light transmittance and yield of plantain juice by 45.7% and 10.0%, respectively ( Table 2). In comparison to the AfumExoPG28A or endo-PG alone, the enzyme combinations showed a more significant effect in pectin degradation. This synergy effect may be caused by targeting the terminal groups of the pectic molecule by exo-PG, and randomly cleaving the inner α-1,4 linkages by endo-PG [3]. Therefore, although endo-PG plays a more important role than the exo-PG in pectin hydrolysis, the presence of exo-PG is necessary for complete degradation.
Tropical and subtropical fruits rich in pectin perish quickly, so processing them into juice not only avoids waste but also improves their added value [44]. Some exo-PGs were obtained by purification or heterologous expression, and their application in the processing of tropical and subtropical fruits has also been verified (Table 3). For example, the exo-PG TePG28a from T. leycettanus increased the light transmittance of grape juice by 34% [25]; two exo-PGs from A. niger [39] and P. janthinellum [36] increased the light transmittance of orange juice by 27% and 35%, respectively, and the exo-PG from Zygoascus hellenicus [35] increased the light transmittance of tangerine, orange, and grapefruit juices by 3.51%, 4.36%, and 8.04%, respectively. Although the exo-PGs reported above have good potential application value for producing some tropical and subtropical fruit juices, on the whole, the optimal pH of the enzyme was close to the pH value of the fruit pulp, improving the effects of the enzymes.
In this study, a novel exo-PG (AfumExoPG28A) from A. fumigatus Af293 was heterologously expressed and characterized. The recombinant protein exhibited the highest activity at 55°C and pH 4.0, while also showing activity and stability in a broad pH range. Moreover, AfumExoPG28A demonstrated excellent performance in  juice extraction by increasing the juice yield and light transmittance of plantain pulp. Thus, AfumExoPG28A could be considered as a good candidate enzyme for acidic juice production. This study enriches the existing literature on exo-PGs and provides a theoretical reference for the application of enzymes of this type.